I used, for my Botryosphaeriaceae isolates, both CTAB and the Qiagen plant kit, and I had no problems.

Just a question: how do you prepare your fungal samples before the DNA extraction?

It should work nicely, However you can use CTAB method, which i usually use for DNA extraction from diverse plant pathogens and has not shown any problem. Buffer contains (100mM TrisHCl, 20mM EDTA; Both at 8.0pH; 1.4M Nacl; 2% CTAB; and 1% PVP + 0.5% betamerceptethanol). Incubate samples at 650C for 1 hr and then add equal vol of Ch:Isoamyl (24:1) centifuge and add Isopropanol and keep for overnight. then PPt. the DNA using 70% ethanol.

Try this hope you will be able to isolate a good quality DNA

Hi Meamiche,

We use the Qiagen kit according to the plant protocol and CTAB on a regular basis for fungal DNA extraction. Usually just scrape the mycelium from the culture grown in a petri dish (can also use for liquid grown culture but usually necessary to get rid of excess liquid using for example freeze drying).The plant protocol from the Wizard kit should give reliable extraction as I've used several of their other kits and are comparable to similar kits from other suppliers. The above CTAB also works really well on most fungi I've used it for.

Thank you every body, all of you were so helpful for me, things are more clear for me now.

Andrea Ucello, i had already extract DNA from Botrytis cinerea and i used SDS protocol, it worked very well. as an answer to your question, i hesitate between liquid culturing in PDB during five days Under agitation or scarping colonies directly from PDA plates as Eduard venter suggested.

Dear Meamiche Hayet, I had some problems with PDB liquid cultures, as Eduard Venter well explained.

The easyest way, according to my (poor) experience, is to scrap mycelium from colonies, dry-freeze it with a micropestle and run CTAB protocol (ask me for it, if you need it).

I had excellent results both in terms of quality and quantity. Last but not least, this protocol in cheaper than most of commercial kits you can buy.

Good luck for your research!

I have used this technique to filamentous fungi DNA extraction. You can find in this paper the technique:

A. K. Casali, L. Goulart, L. K. Rosa e Silva et al., “Molecular

typing of clinical and environmental Cryptococcus neoformans

isolates in the Brazilian state Rio Grande do Sul,” FEMS

Yeast Research, vol. 3, no. 4, pp. 405–415, 2003.

It is very easy and works very well.

Good luck!

Fernanda

Hi Meamiche,

My name is Ben and I work in Promega Technical Services.

While we have not developed a protocol specific for your sample type, there are several publications that cite the use of the Wizard Genomic Kit with fungal samples. The primary challenge with fungal samples is with the lysis step and the references describe the modifications made to successfully lyse the samples. In one of reference, the authors sonicated and ground the mycelia in liquid nitrogen prior to adding the Nuclei Lysis Solution. In another reference, mycelia were ground in liquid nitrogen and lyophilized prior to adding a modified lysis buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM EDTA (pH 8.0), 3% SDS and 1% mercaptoethanol. Other comments and links to these publications can be found at the following link: http://www.promega.com/products/pm/sample-types-processed/wizard-genomic-dna-purification-kit-sample-types#fungus

I or one of my colleagues would be happy to discuss this in further detail if you would like.

Please see this page http://www.promega.com/support/customer-and-technical-support/ for contact information.

i totally agree with you Andrea Uccello, naturally i ask you for CTAB protocol you've used ton compare it with mine. thank you very much for your interest.

Here it is!

It would be a pleasure to me if I was helpful to you!

Dear Andrea, I made ​​you my apologies, my Researchgate account has been blocked for some time. thank you very much for your help and I promise to keep you informed of the results obtained with your protocol. Thank you a thousand times.

Dear Ben Shmidt, i never thought to receive a response from Promega itself. what a nice surprise. I thank you for all your valuable advice, as well as useful links. thank you very much. As i tell Andrea i will inform you about results obtained.

Dear Meamiche, it would be a pleasure to me if I was helpful to you. About your troubles with RG, nevermind, it happens with social networks :)

Dear Meamiche,

I hope you are well. My name is Robert Deyes and like Ben I work with Promega Technical Services. i am writing just to see how things have gone with your purification from mycelia and whether the above tips have helped improve your results.

Many thanks,

Robert

Robert Deyes

Technical Services Scientist

Promega Corporation

Tel 1 800 356 9526 Ext 1366 (USA)

Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)

http://www.promega.com

email: [email protected]

Hi Meamiche,

I've extracted good quality genomic DNA from Mycosphaerella graminicola cultures using the Nucelon Phytopure kit:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/25006720

If you make a spore suspension with sterile water, then spin it down in an eppendorf to pellet the cells and remove the supernatent you can freeze the pellet at -80C for storage and/or proceed with the DNA extraction following the Phytopure kits instructions. I've isolates good quality, high yielding genomic DNA this way for use in qPCR reactions and other downstream processes. IThis kit has been particularly good for extracting genomic DNA from Mycosphaerella species that are difficult to obtain high yields good quality DNA extraction from (e.g. Ramularia collo-cygni).

Hope it helps?

G

Hello Meamiche Hayet, try this. Before the Nuclei Lysis step, use physical lysis with liquid nitrogen and mortar. This technique is useful for both liquid and solid culture.